Ethanol Potentiation of GABA Release onto Ventral Tegmental Area Dopamine Neurons

Activation of ventral tegmental area (VTA)-dopaminergic (DA) neurons by ethanol has been implicated in the rewarding and reinforcing actions of ethanol. GABAergic transmission is thought to play an important role in regulating the activity of DA neurons. We have reported previously that ethanol enhances GABA release onto VTA-DA neurons in a brain slice preparation. Because intraterminal Ca levels regulate neurotransmitter release, we investigated the roles of Ca dependent mechanisms in ethanol-induced enhancement of GABA release. Acute ethanol enhanced miniature inhibitory postsynaptic current (mIPSC) frequency in the presence of the nonspecific voltage-gated Ca channel inhibitor, cadmium chloride, even though basal mIPSC frequency was reduced by cadmium. Conversely, the inositol-1,4,5triphosphate receptor inhibitor, 2-aminoethoxydiphenylborane, and the sarco/endoplasmic reticulum Ca ATPase pump inhibitor, cyclopiazonic acid, eliminated the ethanol enhancement of mIPSC frequency. Recent studies suggest that the G protein-coupled receptor, 5-hydroxytryptamine (5-HT) 2C, may modulate GABA release in the VTA. Thus, we also investigated the role of 5-HT2C receptors in ethanol enhancement of GABAergic transmission. Application of 5-HT and the 5-HT2C receptor agonist, Ro-60-0175 [( S)-6-chloro-5-fluoro-methyl-1H-indole-1-ethanamine fumarate], alone enhanced mIPSC frequency of which the latter was abolished by the 5-HT2C receptor antagonist, SB200646 [N-(1-methyl-5-indoyl)-N-(3pyridyl)urea hydrochloride], and substantially diminished by cyclopiazonic acid. Furthermore, SB200646 abolished the ethanol-induced increase in mIPSC frequency and had no effect on basal mIPSC frequency. These observations suggest that an increase in Ca release from intracellular stores via 5-HT2C receptor activation is involved in the ethanol-induced enhancement of GABA release onto VTA-DA neurons. The mesolimbic system bidirectionally encodes information related to positively and negatively reinforcing stimuli and, with conditioning, will adapt its output to such stimuli in a manner that encodes the error in reward prediction (Schultz et al., 1997). This capability is primarily processed via ventral tegmental area dopaminergic (VTA-DA) neurons, which originate in the midbrain nucleus A10 and project to the nucleus accumbens (NAc), prefrontal cortex, basolateral amygdala, and other corticolimbic structures (Albanese and Minciacchi, 1983; Oades and Halliday, 1987). Natural positive reinforcers activate VTA-DA neurons to release DA onto these structures, of which the medium spiny neurons of the NAc constitute the primary VTA-DA target. Most drugs of abuse pharmacologically activate VTA-DA neurons via a vaThis work was supported in part by the F. M. Jones and H. L. Bruce Endowed Graduate Fellowship; the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grant 1F31AA017020]; and the National Institutes of Health [Grants RO1AA14874, RO1AA1516]. Parts of this work were previously presented at the following conferences: Theile JW, Morikawa H, Gonzales RA, and Morrisett RA (2008) Role of extracellular and intracellular calcium in ethanol enhancement of GABAergic transmission onto VTA-dopamine neurons. Joint Scientific Meeting of the Research Society on Alcoholism and the International Society for Biomedical Research on Alcoholism; 2008 Jun 27-Jul 2, 2008. Washington, D.C.; and Theile JW, Morikawa H, Gonzales RA, and Morrisett RA (2008) Role of 5-HT2C receptors and calcium in ethanol enhancement of GABAergic transmission onto VTA-dopamine neurons. Society for Neuroscience; 2008 Nov 1519; Washington, D.C. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.147793. ABBREVIATIONS: VTA, ventral tegmental area; DA, dopaminergic; NAc, nucleus accumbens; DAMGO, [D-Ala, N-Me-Phe, Gly-ol]-en-kephalin; 5-HT, 5-hydroxytryptamine; 5-HT2CR, 5-HT2C receptor; IP3, inositol-1,4,5triphosphate; aCSF, artificial cerebrospinal fluid; mIPSC, miniature inhibitory postsynaptic current; TTX, tetrodotoxin; 2-APB, 2-aminoethoxydiphenylborane; Ro-60-0175, ( S)-6-chloro-5-fluoro-methyl-1H-indole-1-ethanamine fumarate; SB200646, N-(1-methyl-5-indoyl)-N-(3-pyridyl)urea hydrochloride; ANOVA, analysis of variance; K-S, KolmogorovSmirnov; VGCC, voltage-gated Ca channel; IP3R, IP3 receptor; SERCA, sarco(endo)plasmic reticulum Ca 2 ATPase; CPA, cyclopiazonic acid; sIPSC, spontaneous IPSC; MOR, -opioid receptor. 0022-3565/09/3292-625–633$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 329, No. 2 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 147793/3463974 JPET 329:625–633, 2009 Printed in U.S.A. 625 at A PE T Jornals on Jauary 9, 2018 jpet.asjournals.org D ow nladed from riety of mechanisms, culminating in aberrant release of DA onto these targets, and such pathological activation of the mesolimbic pathway is considered a primary step in the development and expression of drug dependence and addiction. Thus, acute exposure to ethanol increases dopamine levels in the NAc (Weiss et al., 1993). Furthermore, chronic exposure to ethanol has been reported to down-regulate VTA-DA release, and this state of dopaminergic hypofunction is thought to contribute to alcohol craving in dependent individuals (Rossetti et al., 1992). Although the mechanisms by which ethanol activates VTA-DA neurons to enhance DA output to the NAc are not entirely clear, evidence suggests a direct excitatory effect of ethanol on DA neurons in the VTA, either though inhibition of delayed-rectifying K channels or modulation of the h-current that underlies the basal pacemaker function of VTA neurons (Gessa et al., 1985; Brodie et al., 1999; Okamoto et al., 2006; Koyama et al., 2007). Dopamine output from the VTA is regulated by both local GABAergic interneurons and GABA containing afferents from the NAc and ventral pallidum (Walaas and Fonnum, 1980; Grace and Onn, 1989; Johnson and North, 1992a). It is remarkable that our understanding of the effects of ethanol on GABAergic transmission in the mesolimbic DA system is not well defined and is currently in dispute. We have reported that pharmacologically relevant concentrations of ethanol enhance action potential dependent and independent GABA release onto VTA-DA neurons (Theile et al., 2008). On the other hand, Ye and colleagues (Xiao and Ye, 2008) recently demonstrated that ethanol decreases action potentialdependent GABA release onto VTA-DA neurons in a manner reversed by the -opioid receptor agonist, DAMGO. This discrepancy is particularly important because one of the few currently Food and Drug Administration-approved agents for alcohol craving, the -opiate antagonist, naltrexone, is thought to directly modulate VTA-GABAergic neuron activity and thus indirectly affect VTA-DA neuron firing (Franklin, 1995). Naltrexone has been demonstrated to block the ethanol-induced increase in NAc dopamine levels in animal models (Gonzales and Weiss, 1998). Thus, in this report, we have focused our work to uncover VTA-GABAergic regulatory mechanisms that may indirectly modulate ethanol effects on VTA-DA output. One candidate regulatory pathway to the VTA consists of serotoninergic afferents from the midbrain raphe nuclei that innervate both DA and non-DA (GABAergic) neurons of the VTA (Hervé et al., 1987). Electrophysiological evidence indicates that serotonin [5-hydroxytryptamine (5-HT)] reuptake blockade inhibits VTA-DA firing, suggesting that a predominant action of 5-HT in the VTA is inhibitory and may involve activation of GABAergic neurons (Di Mascio et al., 1998). This contention is supported by the observations that: 1) 5-HT2C receptor (5-HT2CR) activation also inhibits VTA-DA neuron firing (Di Matteo et al., 2000), 2) 5-HT2CR mRNA is expressed in GABAergic neurons (Eberle-Wang et al., 1997), and 3) 5-HT2CRs are localized to both synaptic terminals and cell soma of VTA-GABA neurons, although these receptors have also been reported to occur on DA neurons as well (Bubar and Cunningham, 2007). Because 5-HT2CRs facilitate phospholipase C-mediated inositol-1,4,5triphosphate (IP3) accumulation and release of Ca from intracellular stores (Conn et al., 1986), activation of this pathway on GABAergic soma or terminals could very well result in enhancement of GABA release. Activation or inhibition of 5-HT2CRs decreases or increases, respectively, ethanol self-administration by rats (Tomkins et al., 2002). In addition, enhancement of GABA release by ethanol onto cerebellar Purkinje neurons is dependent upon mobilization of Ca from intracellular stores (Kelm et al., 2007). Lastly, no prior reports exist that directly assess whether 5-HT2CR activation regulates GABA release onto VTA-DA neurons. Therefore, another major impetus for this study is to identify the role of 5-HT2CRs and subsequent intracellular Ca mobilization in ethanol facilitation of GABA release onto VTA-DA neurons. Materials and Methods Slice Preparation. All experiments were carried out in accordance with the Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996) and were approved by the University of Texas Institutional Animal Care and Use Committee. Slices used in this study were prepared from male SpragueDawley rats (postnatal days 21–33). Rats were anesthetized with halothane, decapitated, and the brain was rapidly removed and placed in an ice-cold choline-based, oxygenated artificial cerebrospinal fluid (aCSF) containing 110 mM choline Cl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 25 mM dextrose, 7 mM MgSO4, 0.5 mM CaCl2, 11.6 mM sodium ascorbate, and 3.1 mM sodium pyruvate, bubbled with 95% O2/5% CO2 (all chemicals obtained from Sigma-Aldrich, St. Louis, MO). Horizontal midbrain slices (210 m) were prepared using a vibrating slicer (VT1000S; Leica, Wetzlar, Germany). The slices were then maintained at 32°C before electrophysiological recordings for a minimum of 60 min in aCSF containing: 120 mM NaCl, 25 mM NaHCO3, 3.3 mM KCl, 1.23 mM NaH2PO4, 10 mM dextrose, 2.4 mM MgSO4, and 1.8 mM CaCl2, bubbled with 95% O2/5% CO2. Electrophysiological Recordings of VTA-DA Neurons. Individual slices were transferred to a recording chamber and perfused with oxygenated aCSF (30–32°C) at a flow rate of 2 ml/min. Recording aCSF was as described above except it contained 0.9 mM MgSO4 and 2 mM CaCl2. Cells were visualized using IR-DIC optics on an Olympus BX-50WI microscope (Leeds Instruments, Inc., Irving, TX). The VTA was identified as being medial to the medial terminal nucleus of the accessory optic tract and rostral to the oculomotor nerve and the medial lemniscus. The majority of recordings were conducted in the lateral VTA, just medial to the medial terminal nucleus of the accessory optic tract. Whole-cell voltageclamp recordings were used for all experiments; putative DA neurons were identified by the presence of a large hyperpolarizationactivated cationic current ( 200 pA) that was measured immediately after break-in by application of a 1.5-s hyperpolarizing step from 60 to 110 mV (Johnson and North, 1992b). Recording electrodes were made from thin-walled borosilicate glass (TW 150F-4; WPI, Sarasota, FL; 1.5–2.5 M ) and contained 135 mM KCl, 12 mM NaCl, 0.5 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP, and 0.3 mM Tris-GTP, pH 7.3, with KOH (all chemicals obtained from Sigma-Aldrich). Data were collected by an Axon Instruments model 200B amplifier filtered at 1 kHz and digitized at 10 to 20 kHz with a Digidata interface using pClamp version 9.2 and 10.2 (Molecular Devices, Sunnyvale, CA). GABAergic mIPSCs were pharmacologically isolated with kynurenic acid (1 mM) to inhibit -amino-3-hydroxy-5-methyl-4isoxazolepropionic acidand N-methyl-D-aspartate receptor-mediated currents. Tetrodotoxin (TTX; 0.5 M) and eticlopride (250 nM) were included to block Na currents and D2 receptor-mediated currents, respectively. Under these conditions, mIPSCs were inward at a holding potential of 60 mV, and in an initial set of experiments, their identity as GABAeric events was verified by testing for block with picrotoxin or bicuculline (data not shown). After break-in and a stable 10-min baseline (control) recording, drugs were bath-applied 626 Theile et al. at A PE T Jornals on Jauary 9, 2018 jpet.asjournals.org D ow nladed from through the aCSF perfusion line, and a continuous 10to 15-min recording epoch was used to detect changes in mIPSC frequency and amplitude. A 4-min drug wash-on preceded the start of data collection in each treatment condition, and a 12-min washout period followed drug application. The last half (6 min) of the washout period was used in our data analysis. The number of neurons used per each treatment condition is represented as n, with only one neuron used per slice. Eticlopride hydrochloride, 2-aminoethoxydiphenylborane (2-APB), cyclopiazonic acid [(6aR,11aS,11bR)-rel-10-acetyl-2,6,6a,7, 11a,11b-hexahydro-7,7-dimethyl-9H-pyrrolo[1 ,2 :2,3]isoindolo[4,5,6-cd]indol-9-one], bicuculline methiodide, and Ro-60-0175 fumarate were obtained from Tocris Bioscience (Ellisville, MO). Kynurenic acid, SB200646, nicardipine hydrochloride [1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)methyl-2-[methyl(phenylmethyl)amino]-3,5-pyridinedicarboxylic acid ethyl ester hydrochloride], cadmium chloride, serotonin hydrochloride, and picrotoxin were obtained from Sigma-Aldrich. TTX was obtained from Alamone Labs (Jerusalem, Israel). Data Analysis. For mIPSC recordings, quantal events (90 –120 sweeps each condition, 5 s/sweep) were detected using the template mIPSC detection protocol contained within pClampfit (pClamp version 9.2; Molecular Devices, Sunnyvale, CA). Access resistance ranged from 6 to 20 M and was monitored throughout the experiment. Experiments where access resistance changed ( 20% at any time during the experiment) were excluded from this study. To minimize detecting small noise deflections as mIPSCs (false positives), events 10 pA were discarded. Treatment and washout groups were normalized to the baseline (control) frequency or amplitude and represented as a percentage of the control. Averaged values for all data sets are expressed as mean S.E.M. and were compared statistically using paired Student’s t test, unpaired Student’s t test, one-way analysis of variance (ANOVA), and Bonferroni post hoc test where mentioned. The events encompassed in the histogram insets (Fig. 1) were compared using a Kolmogorov-Smirnov (K-S) test. Significant differences were considered as p 0.05 ( ) and p 0.01 ( ).